The present invention relates to a biosensor capable of rapid and simple quantification of a specific component contained in a sample and a method of substrate quantification.
Various biosensors have been proposed in order to readily quantify a specific component of a sample solution without diluting or stirring the sample solution. One example of such biosensors is proposed in the Japanese Laid-Open Patent Publication 2000-39416.
This biosensor is produced by forming an electrode system comprising a working electrode, a counter electrode and a reference electrode on an insulating base plate by screen printing or the like and subsequently forming an enzyme reaction layer comprising an oxidase and an electron mediator on the electrode system.
When a sample solution containing a substrate is dropped on the enzyme reaction layer of the biosensor, dissolution of the enzyme reaction layer takes place to cause a reaction between the enzyme and the substrate, which is accompanied by reduction of the electron mediator. Upon completion of the enzyme reaction, the reduced electron mediator is oxidized electrochemically, and the concentration of the substrate in the sample solution can be determined from the oxidation current value measured in this oxidizing step.
In order to determine the concentration of cholesterol in a sample solution such as blood, plasma or serum, this conventional biosensor includes two kinds of enzymes, that is, cholesterol esterase which hydrolyzes esterified cholesterol and cholesterol oxidase which oxidizes free cholesterol.
However, the measuring sample such as blood, plasma or serum has a pH buffering capacity around neutrality and therefore is not necessarily appropriate in terms of pH for the biosensor comprising cholesterol-oxidizing enzyme and cholesterol esterase.
In view of such problem, an object of the present invention is to provide a biosensor having high response characteristics and high performance by clarifying the appropriate pH and preferable buffers for the biosensor comprising cholesterol-oxidizing enzyme and cholesterol esterase.
A biosensor in accordance with the present invention comprises: an electrically insulating base plate; an electrode system including a working electrode and a counter electrode formed on the base plate; a cover member joined with the base plate to define a sample supply pathway through which a sample solution is introduced from a sample supply port to the electrode system, the sample supply pathway being formed between the cover member and the base plate; and a reagent layer formed in the sample supply pathway, wherein the reagent layer contains a cholesterol-oxidizing enzyme, cholesterol esterase, an electron mediator, and a buffer having a buffering capacity in an acidic pH range.
A method of substrate quantification in accordance with the present invention is a method using a biosensor comprising: an electrically insulating base plate; an electrode system including a working electrode and a counter electrode formed on the base plate; a cover member joined with the base plate to define a sample supply pathway through which a sample solution is introduced from a sample supply port to the electrode system, the sample supply pathway being formed between the cover member and the base plate; and a reagent layer formed in the sample supply pathway, wherein the reagent layer contains a cholesterol-oxidizing enzyme, cholesterol esterase and an electron mediator, and this method comprises: a pretreatment step of mixing a buffer having a buffering capacity in an acidic pH range with the sample solution; a step of supplying the sample solution subjected to the pretreatment step to the biosensor; and a step of determining a substrate contained in the sample solution in the biosensor.
The present invention provides, in another aspect, a measuring system comprising: the above-described biosensor; voltage application means for applying a voltage between the working electrode and the counter electrode; and current detection means for detecting a current flowing between the working electrode and the counter electrode upon application of the voltage.
While the novel features of the invention are set forth particularly in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.